A Novel Method for the Direct Quantification of Gene Transfer Into
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Gene Therapy (1998) 5, 755–760 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt A novel method for the direct quantification of gene transfer into cells using PCR in situ ´ C Catzavelos1, C Ruedy1, AK Stewart2 and I Dube1,2 1Department of Laboratory Medicine, Sunnybrook Health Science Centre; and 2Oncology Gene Therapy Program, The Toronto Hospital, Toronto, Ontario, Canada There are several limitations to current methods for the readily identified by fluorescence microscopy and a high detection of target genes following gene transfer. We sensitivity, specificity and correlation coefficient were dem- report a novel PCR in situ procedure which overcomes onstrated in mixing experiments using varying proportions many of these and permits the direct quantification of gene of known provirus positive and negative cells. The method transfer in individual cells. PCR amplification of a proviral was applied successfully to identify low numbers of gene- specific nucleotide sequence in target cells was followed modified hematopoietic cells in clinical specimens in a trial by in situ hybridization using fluorescent probes com- of retrovirus-mediated gene transfer into blood forming plementary to different regions of the amplicon. Many of stem cells. This approach is simple and reliable, has the the problems previously encountered using in situ PCR, potential for use in a variety of gene therapy applications particularly the generation of false positive results and and may become the method of choice for the assessment extracellular leakage of PCR products, were overcome by of gene transfer efficacy. modifications of existing protocols. Positive cells were Keywords: in situ PCR; retrovirus-mediated gene transfer; hematopoiesis Introduction addition, marker genes may induce changes in the expression of endogenous genes which may alter a wide Gene therapy offers an opportunity to translate advances variety of cellular functions.12 in molecular medicine for the direct benefit of the patient. In situ PCR (ISPCR) permits the intracellular detection At this point in the development of genetic therapies, sig- of PCR products and has the potential to document the nificant emphasis remains on the development of safe proportion of cells modified by gene transfer. This tech- 1,2 and efficient gene delivery systems. Using current tech- nique has been used extensively for viral detection,13–15 niques, the proportion of cells bearing the transferred as well as to detect low abundance mRNA16 and single gene is generally less than 5% of total cells present in copy DNA sequences in situ.17 A number of technical 3 patient material obtained after gene transfer. Thus, sensi- difficulties have been encountered using this method tive and specific detection methods are required for however, the most important being generation of false monitoring gene marked cells in vivo. There are several positive results and leakage of PCR product from target limitations to the current methods used to detect target cells.18–21 4–6 genes. For example, PCR does not permit reliable We report the development of a simple and reliable identification of the transferred gene in specific cells or PCR in situ approach which overcomes many of these quantification of transfer efficacy and Southern analysis limitations. Application of this procedure in a clinical is insufficiently sensitive to detect single copy genes in trial of stem cell marking using a neor construct demon- small cell populations. Fluorescence in situ hybridization strated its utility by identifying gene marked hematopo- 7 (FISH) has been used to assess gene transfer but in most ietic cells. This method has the potential to be applied in gene therapy applications, the target size is usually less a number of gene therapy settings in which the evalu- than 10 kb making the procedure technically difficult, as ation of single copy sequences in individual cells is well as creating the requirement for sophisticated equip- required, and may complement existing technologies. ment for analysis and interpretation. Bi-cistronic con- structs incorporating a marker gene, such as B-galacto- sidase8 or green fluorescent protein9 may be used for the Results detection of gene-modified cells, but the presence of an A Jurkat cell line with and without a stable neor integrant additional exogenous gene may affect expression of the was used to develop the method.22,23 Neor positive and 10 11 therapeutic gene or elicit a host immune response. In negative cells were mixed in equal proportion and slides were prepared by dropping cell suspensions pretreated by exposure to hypotonic buffer and fixed in methanol- r Correspondence: C Catzavelos, Sunnybrook Health Science Centre, Room acetic acid on to glass slides. Several neo specific primer E-433, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5 pairs were evaluated by solution phase PCR and the pair Received 31 July 1997; accepted 13 January 1998 with the most efficient amplification was selected. The Direct quantification of gene transfer with ISPCR C Catzavelos et al 756 PCR product generated was 432 bp. Initially, a protocol samples from individuals enrolled in a clinical trial of involving a single PCR reaction with direct incorporation stem cell gene marking who were undergoing autologous of fluorescein-dUTP into the PCR reaction product was bone marrow transplantation for chemotherapy resistant used. Following a ‘hot start’ procedure to reduce mis- multiple myeloma.24 Post-infusion blood and marrow priming,20 the slides were subjected to 40 PCR cycles. samples as well as PHA/IL2 and EBV-stimulated Thereafter they were washed once for 10 min in 2 × SSC lymphoid cultures were evaluated. Neor-positive cells at 55°C and visualized using fluorescence microscopy. were consistently detected in both peripheral blood and Nonspecific PCR product was detected in the majority of bone marrow samples. There were no ambiguous cells, cells and extensive extracellular leakage was observed. In background or evidence of extracellular leakage.25,26 subsequent experiments, the amount of fluorescein-dUTP in the reaction mixture was decreased, the number of PCR cycles was reduced and the stringency of the post- Discussion hybridization wash increased but nonspecific product formation and extracellular leakage persisted. DNA ISPCR offers a means to detect single copy To address these problems, a two-step approach was sequences in individual cells. There are two approaches used involving an initial PCR reaction with unlabeled in current use. The direct method involves a single in situ primers and subsequent detection by in situ hybridization PCR reaction using labeled nucleotides, while the indirect using two nonoverlapping, fluorescein labeled probes protocol consists of an initial PCR reaction without la- spanning the amplicon. Experiments using 40 amplifi- beled nucleotides, followed by in situ hybridization using cation cycles showed PCR product in the expected per- labeled probes to the PCR products generated. A number centage of neor-positive cells, but extensive extracellular of technical problems have been encountered using this leakage remained a problem. This was markedly reduced technique, the most significant being the generation of by a decrease in the number of PCR cycles from 40 to 15. false positive results.18–21 This has been more commonly An initial post-hybridization wash stringency of 2 × SSC associated with the direct method due to the formation of at 55°C was used but considerable background was nonspecific PCR products from mispriming or products observed. This was eliminated by increasing the post- generated from nonspecific extension of single strand hybridization wash temperature to 62°C (Figure 1a). DNA nicks (end-labeling artifact).18 This creates the Negative controls included pretreatment of slides with requirement for multiple, stringent controls and DNase as well as use of the reaction mixture without Taq additional validation of results particularly when using polymerase or without primers. No positive cells were the direct method. We have attempted both approaches observed (Figure 1b). RNase pretreatment had no effect using multiple controls and shown the indirect method on the results obtained. to be more reliable. This reduces the problem of false As an additional control and to evaluate sensitivity, positive results by allowing visualization of only specific specificity and the correlation coefficient of this method, amplicons in contrast to those generated by mispriming mixing experiments were performed. Provirus-positive or end-labeling. In the direct approach, all PCR products Jurkatt cells were diluted with negative cells in varying are detected due to direct incorporation of labeled proportion (0–100%), subjected to ISPCR as outlined nucleotides. above and scored in a blinded fashion. The experimental Another significant obstacle has been leakage of PCR data is shown in Table 1. The calculated sensitivity and product from target cells.18,19,21 In some studies, false specificity was 96 and 93.7%, respectively. The Pearson positive results have occurred as a result of diffusion of correlation coefficient comparing actual with observed amplicons into nontarget cells.26 In our experiments, this score was 0.996. A graph derived from this data is shown problem was dramatically reduced by decreasing the in Figure 2. number of PCR cycles from 40 to15. No product was vis- This method was subsequently utilized to assess the ualized when the number of cycles was reduced to 10. proportion of gene-modified hematopoietic cells in It is likely that 15 cycles generate sufficient product for detection by in situ hybridization, but not enough to pro- mote extracellular diffusion. In addition, there is also a reduction in the time available for product leakage. Pri- mers generating a product towards the upper limit of the optimum size range for ISPCR27 were used to reduce extracellular diffusion further. We did not find it neces- sary to use other strategies to obviate this problem, such as the use of multiple primer sets, extra-long products, overlapping primers, post-PCR fixation with paraformal- a b dehyde or biotinylated primers.28,29 A post-hybridization wash of 62°C was used.